High performance liquid chromatography (HPLC) is a very common practice in analyzing bioactive materials, drug, toxic materials and metabolites, et al. In practice, a reverse phase high performance liquid chromatographic column is adopted by most people in the art. However, reverse phase high performance liquid chromatographic columns are relatively expensive and have a certain periods of operative life, moreover a reverse phase HPLC column marketed by different manufacturers will have different characteristics, therefore a quick and effective method for evaluating the performance thereof is desired for the operators.
The separating performance of a reverse phase HPLC column is commonly expressed by resolution R, which is dependent on the capacity factor K', separation factor .alpha. and theoretical plate number N. The resolution R can be simply calculated: ##EQU1## The higher the R value, the better the resolution.
At present, the standards used for testing the reverse phase HPLC columns performances are varied between different manufacturers. A composition comprising aromatic compounds such as benzene, naphthalene, toluene, anthracene, methyl benzoate, and the like is the type of standards most commonly used. The compounds contained in this type of standards have the same functionality and only deviate in polarity, consequently they are limited to evaluate the capacity factor, peak symmetry and to calculate the theoretical plate number.
R. M. Smith in his article, entitled "Characterization of Reversed-Phase Liquid Chromatography Columns with Retention Indexes of Standards Based on an Alkyl Aryl Ketone Scale." Anal. Chem., 56, 256 (1984), discloses an improved composition for evaluating the reverse phase HPLC columns comprising different functionality compounds as its components such as p-cresol, nitrobenzene, 2-phenylethanol and toluene. However, the difference in functionality between these constituting components does not have a well designed regularity.
An article by E. J. Kikta, Jr., et al. entitled "Phenones: A Family of Compounds Broadly Applicable to Use as Internal Standards in High-Performance Liquid Chromatography." J. Chromatogr., 138, 41 (1977), discloses another standards for evaluating the reverse phase HPLC columns, which comprises alkylphenones each having various chain length. The alkylphenones all have the same ketone functionality but different polarities due to the various chain lengthes of the alkyl chains.
Therefore, none of the standards for evaluating the performance of the reverse phase HPLC columns comprises compounds each having multiple functionalities, compounds being paired stereo isomers, and compounds being paired positional isomers, which is not only able to be used for evaluating the performance of the reverse phase columns in general aspect but in the aspect of separating stereo- and positional isomers.
The main purpose of the present invention is to provide a composition for evaluating the performance of reverse phase HPLC columns in both the conventional aspect and the aspect of separating stereo- and positional isomers.
Another purpose of the present invention is to provide a method for evaluating the performance of reverse phase HPLC columns.